Phenotypic and Genomic Insights Into Ultraviolet Resistance of Arthrobacter and Pseudarthrobacter Isolated From Desert Soil
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Soil microbial communities are important contributors to the desert ecosystem. Top soil is vital for biological activity but is often exposed to large amounts of DNA damaging ultraviolet (UV) radiation. In this thesis, soil bacteria from the Antarctic Dry Valleys and the Namib Desert were investigated for their resistance and sensitivity to UV radiation. The primary objective of this research was to provide genomic insights into how soil bacteria in arid deserts may survive UV radiation. To achieve this objective, abiotic drivers of the bacterial community were investigated, UV resistant bacteria were identified via a rapid screening technique, and whole genome analysis using comparative genomic approaches and predicted protein tertiary structures was carried out. In the first part of this study, the environmental DNA (eDNA) of each desert was investigated to examine the overall bacterial community. This was done using Illumina based 16S rRNA gene-defined community diversity; for this analysis the V3 and V4 hypervariable regions of the 16S rRNA gene were targeted. Actinobacteria was the most abundant phylum in most of the desert locations. The soil chemistry for the Dry Valleys and the Namib Desert was mostly within the range of published values, except for cation-exchange capacity in the Namib Desert. This range appeared to be influenced by the high concentration of calcium found in the Namib Desert soil. Bacteria isolated from desert soil were exposed to UV radiation using a modified plate drop method for rapid screening of UV resistance or sensitivity. A total of 285 bacterial isolates were tested on solid growth media. All isolates survived exposure to UVA and 35 also survived 10 minutes of 15 W/m2 UVB radiation. In addition, through this method, 16 of the 285 isolates were deemed resistant to 5 W/m2 UVC radiation and 10 were deemed sensitive to 5 W/m2 UVC radiation. Sanger sequencing of the 16S rRNA region using the 27F and 1492R primers identified four genera; Arthrobacter, Pseudarthrobacter, Pseudomonas and Stenotrophomonas, that contained isolates both resistant and sensitive to 5 W/m2 of UVC radiation. As Actinobacteria was the most abundant phylum identified in the 16S rRNA gene-defined communities, the UV resistance and sensitivity of Arthrobacter and Pseudarthrobacter was further investigated at the genomic level. Comparative analyses of the draft genomes of Arthrobacter and Pseudarthrobacter isolated in this study indicate that the isolates were able to reduce nitrate to nitrite, indicating that they may play a role in the nitrogen cycle. The genomes were also predicted to reduce sulphate to sulphite, indicating the isolates may contribute to the sulphur cycle within soil. Computational genome comparative tools such as OrthoANI and in silico DNA-DNA hybridisation (DDH) indicate that all four isolates selected in this study are genetically distinct from the Arthrobacter and Pseudarthrobacter reference genomes and may be new species. Comparative genome analysis of four isolates revealed the two UVC resistant bacteria shared the gene cytochrome P450 (CYP), a gene that was absent from both the UVC sensitive isolates. Analysis of the genome of the UVC sensitive isolates revealed that the CYP gene was absent. It is theorised in this thesis that the presence of CYP may help the UVC resistant isolates identified in this study survive all three types of UV radiation by activating CYP as a DNA repair enzyme. Conversely, several DNA repair genes involved in base excision, nucleotide excision and recombinational repair pathways were present in both the UV resistant and UVC sensitive genomes, as well as several reference genomes. This indicates the presence or absence of the gene products do not have a role in expression of the UV resistant phenotype; however, the expression and function of the gene products may have a role. To infer function of these proteins, the predicted secondary and tertiary structures were compared within the Arthrobacter and Pseudarthrobacter isolates from this study. Protein alignment and analysis of the predicted tertiary structure of the isolates’ UvrABC proteins revealed that, while the UvrA1, UvrA2a and UvrB proteins appeared similar, the UvrC protein for one of the sensitive isolates appeared to be non-functional. Overall, this study has found that Actinobacteria were one of the most abundant phyla in desert soil from Namibia and the Dry Valleys, however, the bacterial community did not appear to be driven by soil chemistry. This study has also produced cultured bacterial isolates that can survive 10 minutes of 5 W/m2 UVC, and therefore represent an important platform for more in-depth studies of UV resistance and sensitivity within Actinobacteria. Comparative genomics showed differences in UV resistance genes between the UVC resistant and UVC sensitive isolates, most notably the CYP gene. Finally, the predicted tertiary protein structures showed that the UvrA and UvrB proteins appeared to be conserved between the isolates, while the UvrC protein appeared to be less conserved, with this protein appearing to be non-functional in one UVC sensitive isolate. The draft genome sequences of these isolates provide a resource for further investigations into Arthrobacter and Pseudarthrobacter and possible physiological attributes that enable their survival in arid desert locations. Furthermore, this study provides further avenues of investigation into UV resistance genes in bacteria isolated from harsh environments.